The major long term objective of this application is to establish a new strategy to inhibit the enzyme telomerase. Telomerase is a promising universal anti-cancer target. Its activity is found in a majority of cancer cells and immortal cell lines, while being absent from a majority of healthy somatic cells. In addition to this correlation there are strong mechanistic reasons why cancer cells require telomerase. The central hypothesis of this project is that molecules which can bind the RNA/DNA duplex formed during telomerase's catalytic cycle will act as inhibitors of the enzyme, and will therefore be potential anti-cancer therapeutics. The postulated mechanism by which these molecules will inhibit the enzyme is either through the prevention of strand dissociation, a key step in telomerase's catalytic cycle, or by the distortion of the duplex substrate, leading to poor catalysis. Our preliminary studies have demonstrated that known RNA/DNA duplex binding molecules are able to inhibit telomerase, and do so in a manner consistent with this inhibition being due to interaction with the RNA/DNA duplex. The specific aims of this work are: 1) Assess known duplex binding molecules such as intercalators for activity as telomerase inhibitors. This assessment will include in-depth kinetic analysis of the mode of inhibition to definitively characterize the mechanism of action of the compounds 2) Use the most successful compounds as the basis of diverse libraries of compounds synthesized using combinatorial chemistry. The purpose of this is to introduce new moieties into the molecule, which will then introduce specific interactions with the unique surrounding telomerase surfaces. 3) Develop high throughput assays, including affinity methods and enzyme assays, which we will use to identify molecules from the combinatorial libraries with the highest affinity for telomerase. These techniques will allow the rapid assessment of large numbers of compounds, allowing a small group of high affinity compounds to be isolated from a large mixture. Combined, these three aims will allow the validation of a new approach for telomerase inhibition and the development of high specificity, high affinity lead inhibitors.